Immunization with attenuated sporozoites (gamma-irradiated or genetically modified) induces sterile immunity that prevents malaria infection. Attenuated sporozoites maintain their ability to infect the hepatocyte but arrest at an early stage of intrahepatocytic development. To develop a sub-unit vaccine based on this model, we need to identify the targets of immunity. Previous studies in cancer research demonstrated that heat shock protein (HSP)-chaperoned peptides derived from tumor cells can induce immune responses against the same tumor. To identify peptides carried by HSPs for presentation to the immune system the following experimental approaches are used: 1. HSP-peptide complexes are purified from infected hepatocytes followed by peptide elution and identification by mass spectrometry 2. In-vitro binding studies: purified HSP from uninfected hepatocyte are incubated with a peptide mixture obtained from sporozoite. Bound peptides are eluted from the HSP and analyzed by mass spectrometry. The corresponding proteins will be used to identify orthologs expressed by human parasites. Protein that uniquely expressed during the sporozoite-liver stage developmental stages of the parasites and have orthologs expressed by human parasites will be used to immunize mice followed by challenge with wild type sporozoites to identify protective HSP-peptide complexes. In parallel to the effort to identify new vaccine targets, we have been focusing on identifying biomarkers of immunity to liver-stage parasites. Immunization with attenuated sporozoites (-irradiated) and, more recently, chemoprophylaxis vaccination (CVac) regimens induce immunity that prevents malaria infection. Both vaccine approaches induce CD8+T cells and interferon- responses that have been used as markers of protection. Because earlier trials of both approaches induced sterile immunity in all vaccinated individuals, and all had high IFN-, it has not been possible to assess whether IFN is part of the protective mechanism or a surrogate marker of immunity. A recent CVac trial lead by Patrick Duffy resulted in 3 response phenotypes: sterile protection, delayed time to blood stage parasitemia and no protection. This unique pattern of response to CVac provide the opportunity to identify biomarkers of protective immunity.